Protein ubiquitination is modulated by O-GlcNAc glycosylation

• Published in: The FASEB journal 2008-08, Vol.22 (8), p.2901-2911
• Main Authors: Guinez, Céline, Mir, Anne-Marie, Dehennaut, Vanessa, Cacan, René, Harduin-Lepers, Anne, Michalski, Jean-Claude, Lefebvre, Tony
• Format: Article
• Language: English
• Subjects: Acetylglucosamine - metabolism; Base Sequence; Biochemistry, Molecular Biology; Cell Line; Cell Survival; Glycosylation; heat shock; Heat-Shock Response; HeLa Cells; HSP70 Heat-Shock Proteins - metabolism; Humans; Life Sciences; N-Acetylglucosaminyltransferases - antagonists & inhibitors; N-Acetylglucosaminyltransferases - genetics; N-Acetylglucosaminyltransferases - metabolism; OGT RNA interference; proteasomal degradation; Proteasome Endopeptidase Complex - metabolism; Protein Processing, Post-Translational; Proteins - chemistry; Proteins - metabolism; RNA Interference; RNA, Small Interfering - genetics; Ubiquitin-Activating Enzymes - metabolism; Ubiquitination
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fj.07-102509

During the past two decades, O-GlcNAc modification of cytosolic and nuclear proteins has been intensively studied. Nevertheless, the function of this post-translational modification remains unclear. It has been recently speculated that O-GlcNAc could act as a protective signal against proteasomal degradation, both by modifying target substrates and/or by inhibiting the proteasome itself. In this work, we have investigated the putative relation between O-GlcNAc and the ubiquitin pathway. First, we showed that the level of both modifications increased rapidly after thermal stress but, unlike ubiquitinated proteins, O-GlcNAc-modified proteins failed to be stabilized by inhibiting proteasome function. Increasing O-GlcNAc levels, using glucosamine or PUGNAc, enhanced ubiquitination. Inversely, when O-GlcNAc levels were reduced, using forskolin or glucose deprivation, ubiquitination decreased. Targeted-RNA interference of O-GlcNAc transferase also reduced ubiquitination and moreover halved cell thermotolerance. Finally, we demonstrated that the ubiquitin-activating enzyme E1 was O-GlcNAc modified and that its glycosylation and its interaction with Hsp70 varied according to the conditions of cell culture. Altogether, these results show that O-GlcNAc and ubiquitin are not strictly antagonistic post-translational modifications, but rather that the former might regulate the latter, and also suggest that E1 could be one of the common links between the two pathways. --Guinez, C., Mir, A.-M., Dehennaut, V., Cacan, R., Harduin-Lepers, A., Michalski, J.-C., Lefebvre, T. Protein ubiquitination is modulated by O-GlcNAc glycosylation.